Cytochrome P450 enzymes are a heme-containing family that play central roles in oxidative, peroxidative and reductive metabolism of numerous endogenous and exogenous compounds, including many pharmaceutical agents. Substances known to be metabolized by P450 enzymes include steroids, bile acids, fatty acids, prostaglandins, leukotrienes, biogenic amines, retinoids, lipid hydroperoxides, phytoalexins, pharmaceuticals, environmental chemicals and pollutants. P450 substrates also include natural plant products involved in flavor, odor and flower color. Many P450 enzymes also have functions in maintaining steady-state levels of endogenous ligands involved in ligand-modulated transcription of genes effecting growth, apoptosis, differentiation, cellular homeostasis, and neuroendocrine functions. The metabolism of foreign chemicals by P450 enzymes can produce toxic metabolites, some of which have been implicated as agents responsible for birth defects and tumor initiation and progression.
The P450 gene superfamily is likely to have evolved from an ancestral gene present before the prokaryote/eukaryote divergence. The number of individual P450 genes in any mammalian species is estimated at 60 to 200. The cytochrome P450 (CYP) 3A subfamily is unique in that it is present in large amounts in human liver microsomes, and there are many forms in the subfamily. Several human cDNAs encoding CYP3A proteins have been identified. The most important of these are CYP3A4, CYP3A5 and CYP3A7. CYP3A4 and CYP3A7 genes are 87% homologous by amino acid and 95% homologous by nucleotide sequence, while CYP3A4 and CYP3A5 are only 88% homologous in the coding region. CYP3A4 and CYP3A7 are 91% homologous in the 5′-flanking sequences, differing by the presence of a unique P450NF specific element (NFSE) and a P450HFLa specific element (HFLaSE), respectively (Hashimoto et al, 1993).
Genetic polymorphisms of cytochrome P450 enzymes result in subpopulations of individuals that are distinct in their ability to perform particular drug biotransformation reactions. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to the majority of humans may cause intolerable side-effects in an individual suffering from a defect in a cytochrome P450 enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of the lack of a particular cytochrome P450 enzyme required for conversion of the drug to a metabolically active form. Accordingly, it is important for both drug development and clinical use to screen drugs to determine which cytochrome P450 enzymes are required for activation and/or detoxification of the drug.
It is also important to identify those individuals who are deficient in a particular P450 enzyme. This type of information has been used to advantage in the past for developing genetic assays that predict phenotype and thus predict an individual's ability to metabolize a given drug. Information such as this would be of particular value in determining the likely side effects and therapeutic failures of various drugs and routine phenotyping could be recommended for certain categories of patients.
The CYP3A subclass catalyzes a remarkable number of oxidation reactions of clinically important drugs such as quinidine, warfarin, erythromycin, cyclosporin A, midazolam, lidocain, nifedipine, and dapsone. Current estimates are that more than 60% of clinically used drugs are metabolized by the CYP3A4 enzyme, including such major drug classes as calcium channel blockers, immunosuppressors, macrolide antibiotics and anticancer drugs, see Brian et al., 1990, Biochemistry, vol. 29, pages 11280-11292.
Expression profiles for each member of this family varies significantly. CYP3A4 is expressed in all adult human liver and intestine, accounting for more than 50% of total P450 in both organs. Expression is inducible in vivo and in vitro by numerous compounds such as rifampicin, barbiturates and glucocorticoids. In kidney, CYP3A4 is expressed polymorphically. CYP3A4 expression is sex-influenced, as females have 24% greater expression than males. Substantial inter-individual variation in the metabolism of specific compounds by CYP3A4 has been reported (Kleinbloesem et al., Biochemical Pharmacology, 1984, vol. 33, pages 3721-3724. U.S. Pat. No. 6,174,684 to Rebbeck et al. discloses a CYP3A4 variant associated with a heightened risk of developing or having prostate cancer and a decreased risk for developing treatment-related leukemias. The polymorphism disclosed by Rebbeck et al. is an A to G transition in the promoter region of the CYP 3A4 gene which is thought to alter the nifedipine-specific binding element located 287 to 296 bases 5′ to the CYP 3A4 transcription start site. The genotype associated with this variant is believed to increase the production of potentially DNA damaging reactive intermediates upon patient exposure to an epipodophyllotoxin. CYP3A5 is detected in 10-30% of Caucasian adult livers, and expressed constitutively in adult kidney. CYP3A5 expression does not appear to be sex-influenced and only moderately inducible by xenobiotics both in vivo and in vitro. CYP3A7 is expressed in fetal liver but only in 25% of adult livers. Molecular mechanisms responsible for the developmentally specific expression of CYP3A's are unknown.
Another supergene family of metabolic enzymes is the glutathione S-transferase (GST) superfamily. These enzymes play an important role in the cellular enzymatic protection against the cytotoxic and mutagenic effects of electrophiles. Thus, GST alleles associated with impaired detoxification will confer an increased susceptibility to a wide range of diseases. In particular, GST genotypes have been associated with an increased susceptibility in diseases associated with oxidative stress. One of the best characterized of such associations is the null mutation in the mu class GSTM1 gene. GSTM1 is polymorphic due to large deletions in the structural gene. The null GSTM1 genotype is clearly associated with bladder cancer and lung cancer, and possibly associated with colorectal, hepatocellular, gastric, esophageal, head and neck as well as cutaneous cancer. There is considerable evidence that the combination of the GSTM1 null genotype in combination with the cytochrome P450 1A1 rare alleles confers a highly increased risk of developing lung cancers in smokers. The GSTM1 null genotype has also been found to be significantly associated with an increased risk of developing postmenopausal breast cancer as described by Helzlsouer et al., J. Natl. Cancer Inst., 1998, vol. 90, pages 512-518.
Women have a 15 percent lifetime risk of developing breast cancer. Approximately 10 to 15 percent of all breast cancers are familial, and approximately 33 percent of these may be linked to genetic mutations. Available treatment options include (1) surgery, (2) radiation therapy, (3) chemotherapy, and (4) hormone manipulation. Hundreds of thousands of women are currently undergoing local, as well as systemic, treatment for their breast cancer. Each treatment has its risks and side effects. The risks and side effects of chemotherapy can be substantially reduced and the likelihood of a successful outcome can be increased if the chemotherapeutic regimen selected is tailored to the individual patient. Two drugs commonly used in the treatment of breast cancer are cyclophosphamide and carmustine (BCNU).
Cyclophosphamide is a nitrogen mustard derivative, polyfunctional alkylating agent which is bioconverted from an inert prodrug to an active DNA alkylating agent by the oxidative cytochromes of the liver of which CYP 3A4 and 3A5 are the principle enzymes. Thus, patients with lower levels of either enzyme will produce less of the active forms of cyclophosphamide when given the same dose over the same time as a patient who has normal levels of these activating enzymes. Cyclophospamide functions to interfere with DNA replication and transcription of RNA, ultimately resulting in the disruption of nucleic acid function. The drug also exhibits potent immunosuppressive activity and phosphorylating properties that enhance its cytotoxicity. In the treatment of breast cancer, cyclophosphamide used alone has been reported to produce objective responses in about 35% of patients. Used in combination regimens, objective responses have been reported in up to 90% of patients, and cyclophosphamide-containing combinations are believed by some experts to be the treatment of choice.
BCNU is a nitrosourea with a broad spectrum of activity. It is a classic alkylating agent, but also inhibits DNA repair by isocyanate formation. BCNU is used alone or as a component of various chemotherapy regimens in the treatment of primary or metastatic tumors. BCNU is a highly toxic drug with a low therapeutic index, thus a therapeutic response is unlikely without some evidence of toxicity. The primary toxicities are pulmonary toxicity and hepatic dysfunction which appear to be dose related. Patients receiving cumulative doses exceeding 1400 mg/m2 are at substantially higher risk than patients receiving lower cumulative doses. Thus, any means of screening prospective cancer patients for factors which can effect the dosing of BCNU is of great importance in effectively designing chemotherapy regimens that include BCNU to enhance the clinical outcome while minimizing adverse effects.
Cisplatin is a bifunctional alkylating agent that binds to DNA and inhibits DNA synthesis. The drug produces predominately DNA interstrand crosslinks with some intrastrand crosslinks resulting from the formation of adducts between activated platinum complexes of the drug. Interstrand crosslinking appears to correlate well with the cytotoxicity of the drug. Cisplatin is used to treat a wide variety of neoplasms and is often used as a component of combination chemotherapeutic regimens because of its relative lack of hematologic toxicity. Cisplatin is a highly toxic drug with a low therapeutic index. While hematologic toxicities such as thrombocytopenia and leukopenia are the major dose-limiting adverse effects of cisplatin therapy, other dose-limiting adverse effects including nephrotoxicity, ototoxicity, neurotoxicity, and emesis are frequently seen. These adverse effects are potentiated in patients receiving other antineoplastic agents or drugs with nephorotoxic or ototoxic effects, such as aminoglycoside antibiotics.
Since the rates of metabolism of drugs and toxins can depend on the amounts and kinds of P450s expressed in a tissue, variation in biological response may be determined by the profile of expression of P450s in each person. As noted above, this variation in response may significantly influence the outcome of treating breast cancer patients with different antineoplastic drugs. Analysis of genetic polymorphisms that lead to altered expression and enzyme activity of these metabolic enzymes are therefore of interest.